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==Methods==
==Methods==
There are several methods available for mapping antibody epitopes on target antigens:
There are several methods available for mapping antibody epitopes on target antigens:
* '''[[x-ray crystallography|X-ray co-crystallography]].''' This technique has historically been regarded as the gold-standard approach for epitope mapping because it allows direct visualization of the interaction between the antigen and antibody. However, this approach is technically challenging, time-consuming, and expensive, and it requires large amounts of purified protein. Moreover, not all proteins are amenable to crystallization.<ref>{{Cite journal|last=Abbott WM, Damschroder MM, Lowe DC|first=|date=2014|title=Current approaches to fine mapping of antigen–antibody interactions|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107663/|journal=Immunology|volume=142|pages=526–535.|via=}}</ref>
* '''[[x-ray crystallography|X-ray co-crystallography]].''' This technique has historically been regarded as the gold-standard approach for epitope mapping because it allows direct visualization of the interaction between the antigen and antibody. However, this approach is technically challenging, time-consuming, and expensive, and it requires large amounts of purified protein. Moreover, not all proteins are amenable to crystallization.<ref>{{Cite journal|last=Abbott WM, Damschroder MM, Lowe DC|first=|date=2014|title=Current approaches to fine mapping of antigen–antibody interactions|journal=Immunology|volume=142|issue=4|pages=526–535|pmc=4107663|pmid=24635566|doi=10.1111/imm.12284}}</ref>
* '''[[Peptide microarray|Array]]-based [[peptide|oligo-peptide]] scanning.''' Also known as overlapping peptide scan or [[pepscan|pepscan analysis]], this technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein, and tests for their ability to bind the antibody of interest. This method is fast, relatively inexpensive, and specifically suited to profile epitopes for large numbers of candidate antibodies against a defined target.<ref name=":1">{{cite journal |last1=Gaseitsiwe |first1=S. |last2=Valentini |first2=D. |last3=Mahdavifar |first3=S. |last4=Reilly |first4=M. |last5=Ehrnst |first5=A. |last6=Maeurer |first6=M. |title=Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401 |journal=Clinical and Vaccine Immunology |volume=17 |issue=1 |pages=168–75 |year=2010 |pmid=19864486 |pmc=2812096 |doi=10.1128/CVI.00208-09 }}</ref><ref>{{cite journal |last1=Linnebacher |first1=Michael |last2=Lorenz |first2=Peter |last3=Koy |first3=Cornelia |last4=Jahnke |first4=Annika |last5=Born |first5=Nadine |last6=Steinbeck |first6=Felix |last7=Wollbold |first7=Johannes |last8=Latzkow |first8=Tobias |last9=Thiesen |first9=Hans-Jürgen |last10=Glocker |first10=Michael O. |title=Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples |journal=Analytical and Bioanalytical Chemistry |volume=403 |issue=1 |pages=227–38 |year=2012 |pmid=22349330 |doi=10.1007/s00216-012-5781-5 }}</ref> The epitope mapping resolution depends on the number of overlapping peptides that are used. The main disadvantage of this approach is that it cannot generally be used to obtain conformational epitopes, which are the most relevant epitope type for human therapeutic mAbs. However, one study<ref>{{cite journal|last1=Cragg|first1=M. S.|year=2011|title=CD20 antibodies: doing the time warp|journal=Blood|volume=118|issue=2|pages=219–20|doi=10.1182/blood-2011-04-346700|pmid=21757627}}</ref> rarely mapped discontinuous epitopes on [[CD20]] using array-based oligo-peptide scanning, by combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (e.g., by using CLIPS scaffolds<ref>{{cite journal |last1=Timmerman |first1=P. |last2=Puijk |first2=W. C. |last3=Boshuizen |first3=R. S. |last4=Dijken |first4=P.van |last5=Slootstra |first5=J. W. |last6=Beurskens |first6=F. J. |last7=Parren |first7=P.W. H.I. |last8=Huber |first8=A. |last9=Bachmann |first9=M. F. |last10=Meloen |first10=R. H. |title=Functional Reconstruction of Structurally Complex Epitopes using CLIPS™ Technology |journal=The Open Vaccine Journal |volume=2 |issue=1 |year=2009 |pages=56–67 |doi=10.2174/1875035400902010056 }}</ref>).
* '''[[Peptide microarray|Array]]-based [[peptide|oligo-peptide]] scanning.''' Also known as overlapping peptide scan or [[pepscan|pepscan analysis]], this technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein, and tests for their ability to bind the antibody of interest. This method is fast, relatively inexpensive, and specifically suited to profile epitopes for large numbers of candidate antibodies against a defined target.<ref name=":1">{{cite journal |last1=Gaseitsiwe |first1=S. |last2=Valentini |first2=D. |last3=Mahdavifar |first3=S. |last4=Reilly |first4=M. |last5=Ehrnst |first5=A. |last6=Maeurer |first6=M. |title=Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401 |journal=Clinical and Vaccine Immunology |volume=17 |issue=1 |pages=168–75 |year=2010 |pmid=19864486 |pmc=2812096 |doi=10.1128/CVI.00208-09 }}</ref><ref>{{cite journal |last1=Linnebacher |first1=Michael |last2=Lorenz |first2=Peter |last3=Koy |first3=Cornelia |last4=Jahnke |first4=Annika |last5=Born |first5=Nadine |last6=Steinbeck |first6=Felix |last7=Wollbold |first7=Johannes |last8=Latzkow |first8=Tobias |last9=Thiesen |first9=Hans-Jürgen |last10=Glocker |first10=Michael O. |title=Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples |journal=Analytical and Bioanalytical Chemistry |volume=403 |issue=1 |pages=227–38 |year=2012 |pmid=22349330 |doi=10.1007/s00216-012-5781-5 }}</ref> The epitope mapping resolution depends on the number of overlapping peptides that are used. The main disadvantage of this approach is that it cannot generally be used to obtain conformational epitopes, which are the most relevant epitope type for human therapeutic mAbs. However, one study<ref>{{cite journal|last1=Cragg|first1=M. S.|year=2011|title=CD20 antibodies: doing the time warp|journal=Blood|volume=118|issue=2|pages=219–20|doi=10.1182/blood-2011-04-346700|pmid=21757627}}</ref> rarely mapped discontinuous epitopes on [[CD20]] using array-based oligo-peptide scanning, by combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (e.g., by using CLIPS scaffolds<ref>{{cite journal |last1=Timmerman |first1=P. |last2=Puijk |first2=W. C. |last3=Boshuizen |first3=R. S. |last4=Dijken |first4=P.van |last5=Slootstra |first5=J. W. |last6=Beurskens |first6=F. J. |last7=Parren |first7=P.W. H.I. |last8=Huber |first8=A. |last9=Bachmann |first9=M. F. |last10=Meloen |first10=R. H. |title=Functional Reconstruction of Structurally Complex Epitopes using CLIPS™ Technology |journal=The Open Vaccine Journal |volume=2 |issue=1 |year=2009 |pages=56–67 |doi=10.2174/1875035400902010056 |url=http://dare.uva.nl/personal/pure/en/publications/functional-reconstruction-of-structurally-complex-epitopes-using-clips-technology(ce45bb5a-7823-4872-a0b1-e5e5a99a79e5).html |type=Submitted manuscript }}</ref>).
* '''[[Site-directed mutagenesis]] mapping.''' The molecular biological technique of [[site-directed mutagenesis]] (SDM) can be used to enable epitope mapping. In SDM, systematic [[mutations]] of amino acids are introduced into the sequence of the target protein. Binding of an antibody to each mutated protein is tested to identify the amino acids that comprise the epitope. This technique can be used to map both linear and conformational epitopes, but is labor-intensive and time-consuming, typically limiting analysis to a small number of amino-acid residues.<ref name=":5">{{Cite journal|last=Davidson E, Doranz B|first=|date=2014|title=A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.|url=https://www.ncbi.nlm.nih.gov/pubmed/24854488|journal=Immunology|volume=143|pages=13-20|via=}}</ref>
* '''[[Site-directed mutagenesis]] mapping.''' The molecular biological technique of [[site-directed mutagenesis]] (SDM) can be used to enable epitope mapping. In SDM, systematic [[mutations]] of amino acids are introduced into the sequence of the target protein. Binding of an antibody to each mutated protein is tested to identify the amino acids that comprise the epitope. This technique can be used to map both linear and conformational epitopes, but is labor-intensive and time-consuming, typically limiting analysis to a small number of amino-acid residues.<ref name=":5">{{Cite journal|last=Davidson E, Doranz B|first=|date=2014|title=A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.|journal=Immunology|volume=143|issue=1|pages=13–20|pmid=24854488|pmc=4137951|doi=10.1111/imm.12323}}</ref>
* '''High-throughput shotgun mutagenesis mapping.'''<ref name=":5" /><ref>{{cite web|url=https://s3.amazonaws.com/s3-integral-molecular-product/wp-content/uploads/2017/11/01153232/INTG-Epitope-Mapping-using-Shotgun-Mutagenesis-ilovepdf-compressed-1.pdf|title=Epitope Mapping using Shotgun Mutagenesis Technology|last=|first=|date=2017|website=Integral Molecular|publisher=|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref> The shotgun mutagenesis technique begins with the creation of a [[mutation]] library of the entire target [[antigen]], with each&nbsp;clone&nbsp;containing a unique [[amino acid]] mutation (typically an alanine-to-serine substitution). Hundreds of [[plasmid]] clones from the library are individually arrayed in 384-well micro plates, expressed in mammalian cells, and tested for antibody binding. Amino acids of the target required for antibody binding are identified by a loss of immunoreactivity. These residues are mapped onto structures of the target protein to visualize the epitope.&nbsp;Benefits of high-throughput mutagenesis mapping include: 1) the ability to identify both linear and conformational epitopes, 2) in a fraction of the time required by other methods, 3) the presentation of properly folded and post-translationally modified proteins, and 4) the ability to identify key amino acids that drive the energetic interactions (energetic "hot spots" of the epitope)<ref>{{Cite journal|last=Bogan AA, Thorn KS|first=|date=1998|title=Anatomy of hot spots in protein interfaces|url=|journal=Journal of Molecular Biology|volume=280|pages=1-9|via=}}</ref><ref>{{Cite journal|last=Lo Conte L, Chothia C, Janin J|first=|date=1999|title=The atomic structure of protein-protein recognition sites|url=|journal=Journal of Molecular Biology|volume=285|pages=2177-2198|via=}}</ref>. High-throughput shotgun mutagenesis mapping is a well-validated and widely used approach that has been used to obtain single-amino-acid-resolution epitopes for over 1,000 mAbs,<ref>{{Cite web|url=https://www.integralmolecular.com/epitope-mapping/|title=Epitope Mapping Services|last=|first=|date=|website=Integral Molecular|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref> revealing mechanistic information for protein-drug interactions,<ref>{{Cite web|url=https://s3.amazonaws.com/s3-integral-molecular-product/wp-content/uploads/2017/11/01153107/INTG-Drug-MappingDocking-using-Shotgun-Mutagenesis.pdf|title=Mapping and Docking of GPCR-Drug Interactions using Shotgun Mutagenesis|last=|first=|date=|website=Integral Molecular|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref><ref>{{Cite journal|last=Paes C, Ingalls J, Kampani K, Sulli C, Kakkar E, Murray M, Kotelnikov V, Greene TA, Rucker JB, Doranz BJ|first=|date=2009|title=Atomic-Level Mapping of Antibody Epitopes on a GPCR|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943208/|journal=Journal of the American Chemical Society|volume=131|pages=6952–6954|via=}}</ref> the functional regions of complex membrane proteins, including GPCRs,<ref>{{Cite web|url=https://s3.amazonaws.com/s3-integral-molecular-product/wp-content/uploads/2017/11/01153112/INTG-Ligand-Mapping-using-Shotgun-Mutagenesis.pdf|title=Mapping Functional Regions of GPCRs using Shotgun Mutagenesis|last=|first=|date=2017|website=Integral Molecular|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref><ref>{{Cite journal|last=Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, Breslin PAS, Rucker JB|first=|date=2011|title=Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3101243/|journal=PLOS One|volume=6(5)|pages=e20123|via=}}</ref> target binding information for small molecules,<ref>{{Cite journal|last=Wescott MP, Kufareva I, Paes C, Goodman JR, Thaker Y, Puffer BA, Berdougo E, Rucker JB, Handel TM, Doranz BJ|first=|date=2016|title=Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024644/|journal=PNAS|volume=113(35)|pages=9928–9933|via=}}</ref><ref>{{Cite journal|last=Thomas A, Sulli C, Davidson E, Berdougo E, Phillips M, Puffer BA, Paes C, Doranz BJ, Rucker JB|first=|date=2017|title=The Bitter Taste Receptor TAS2R16 Achieves High Specificity and Accommodates Diverse Glycoside Ligands by using a Two-faced Binding Pocket|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552880/|journal=Scientific Reports|volume=7(1)|pages=7753|via=}}</ref> and epitope information for IP protection purposes.<ref name=":3" /><ref name=":4" />
* '''High-throughput shotgun mutagenesis mapping.'''<ref name=":5" /><ref>{{cite web|url=https://s3.amazonaws.com/s3-integral-molecular-product/wp-content/uploads/2017/11/01153232/INTG-Epitope-Mapping-using-Shotgun-Mutagenesis-ilovepdf-compressed-1.pdf|title=Epitope Mapping using Shotgun Mutagenesis Technology|last=|first=|date=2017|website=Integral Molecular|publisher=|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref> The shotgun mutagenesis technique begins with the creation of a [[mutation]] library of the entire target [[antigen]], with each&nbsp;clone&nbsp;containing a unique [[amino acid]] mutation (typically an alanine-to-serine substitution). Hundreds of [[plasmid]] clones from the library are individually arrayed in 384-well micro plates, expressed in mammalian cells, and tested for antibody binding. Amino acids of the target required for antibody binding are identified by a loss of immunoreactivity. These residues are mapped onto structures of the target protein to visualize the epitope.&nbsp;Benefits of high-throughput mutagenesis mapping include: 1) the ability to identify both linear and conformational epitopes, 2) in a fraction of the time required by other methods, 3) the presentation of properly folded and post-translationally modified proteins, and 4) the ability to identify key amino acids that drive the energetic interactions (energetic "hot spots" of the epitope)<ref>{{Cite journal|last=Bogan AA, Thorn KS|first=|date=1998|title=Anatomy of hot spots in protein interfaces|url=|journal=Journal of Molecular Biology|volume=280|issue=1|pages=1–9|via=|pmid=9653027|doi=10.1006/jmbi.1998.1843}}</ref><ref>{{Cite journal|last=Lo Conte L, Chothia C, Janin J|first=|date=1999|title=The atomic structure of protein-protein recognition sites|url=|journal=Journal of Molecular Biology|volume=285|pages=2177–2198|via=}}</ref>. High-throughput shotgun mutagenesis mapping is a well-validated and widely used approach that has been used to obtain single-amino-acid-resolution epitopes for over 1,000 mAbs,<ref>{{Cite web|url=https://www.integralmolecular.com/epitope-mapping/|title=Epitope Mapping Services|last=|first=|date=|website=Integral Molecular|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref> revealing mechanistic information for protein-drug interactions,<ref>{{Cite web|url=https://s3.amazonaws.com/s3-integral-molecular-product/wp-content/uploads/2017/11/01153107/INTG-Drug-MappingDocking-using-Shotgun-Mutagenesis.pdf|title=Mapping and Docking of GPCR-Drug Interactions using Shotgun Mutagenesis|last=|first=|date=|website=Integral Molecular|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref><ref>{{Cite journal|last=Paes C, Ingalls J, Kampani K, Sulli C, Kakkar E, Murray M, Kotelnikov V, Greene TA, Rucker JB, Doranz BJ|first=|date=2009|title=Atomic-Level Mapping of Antibody Epitopes on a GPCR|journal=Journal of the American Chemical Society|volume=131|issue=20|pages=6952–6954|pmc=2943208|pmid=19453194|doi=10.1021/ja900186n}}</ref> the functional regions of complex membrane proteins, including GPCRs,<ref>{{Cite web|url=https://s3.amazonaws.com/s3-integral-molecular-product/wp-content/uploads/2017/11/01153112/INTG-Ligand-Mapping-using-Shotgun-Mutagenesis.pdf|title=Mapping Functional Regions of GPCRs using Shotgun Mutagenesis|last=|first=|date=2017|website=Integral Molecular|archive-url=|archive-date=|dead-url=|access-date=September 21, 2018}}</ref><ref>{{Cite journal|last=Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, Breslin PAS, Rucker JB|first=|date=2011|title=Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin|journal=PLOS One|volume=6|issue=5|pages=e20123|pmc=3101243|pmid=21629661|doi=10.1371/journal.pone.0020123}}</ref> target binding information for small molecules,<ref>{{Cite journal|last=Wescott MP, Kufareva I, Paes C, Goodman JR, Thaker Y, Puffer BA, Berdougo E, Rucker JB, Handel TM, Doranz BJ|first=|date=2016|title=Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices|journal=PNAS|volume=113|issue=35|pages=9928–9933|pmc=5024644|pmid=27543332|doi=10.1073/pnas.1601278113}}</ref><ref>{{Cite journal|last=Thomas A, Sulli C, Davidson E, Berdougo E, Phillips M, Puffer BA, Paes C, Doranz BJ, Rucker JB|first=|date=2017|title=The Bitter Taste Receptor TAS2R16 Achieves High Specificity and Accommodates Diverse Glycoside Ligands by using a Two-faced Binding Pocket|journal=Scientific Reports|volume=7|issue=1|pages=7753|pmc=5552880|pmid=28798468|doi=10.1038/s41598-017-07256-y}}</ref> and epitope information for IP protection purposes.<ref name=":3" /><ref name=":4" />
* '''[[Hydrogen–deuterium exchange]].''' This method, which is growing in popularity, gives information about the solvent accessibility of various parts of the antigen and antibody, demonstrating reduced solvent accessibility in regions of protein-protein interactions.<ref>{{Cite journal|last=Casina VC, Hu W, Hanby HA, Pickens B, Mayne L, Ostertag E, Kacir S, Siegel DL, Englander SW, Zheng XL|first=|date=2014|title=Autoantibody Epitope Mapping By Hydrogen-Deuterium Exchange Mass Spectrometry at Nearly Single Amino Acid Residue Resolution Reveals Novel Exosites on ADAMTS13 Critical for Substrate Recognition and Mechanism of Autoimmune Thrombotic Thrombocytopenic Purpura|url=http://www.bloodjournal.org/content/124/21/108?sso-checked=true|journal=Blood|volume=124|pages=108|via=}}</ref>
* '''[[Hydrogen–deuterium exchange]].''' This method, which is growing in popularity, gives information about the solvent accessibility of various parts of the antigen and antibody, demonstrating reduced solvent accessibility in regions of protein-protein interactions.<ref>{{Cite journal|last=Casina VC, Hu W, Hanby HA, Pickens B, Mayne L, Ostertag E, Kacir S, Siegel DL, Englander SW, Zheng XL|first=|date=2014|title=Autoantibody Epitope Mapping By Hydrogen-Deuterium Exchange Mass Spectrometry at Nearly Single Amino Acid Residue Resolution Reveals Novel Exosites on ADAMTS13 Critical for Substrate Recognition and Mechanism of Autoimmune Thrombotic Thrombocytopenic Purpura|url=http://www.bloodjournal.org/content/124/21/108?sso-checked=true|journal=Blood|volume=124|pages=108|via=}}</ref>
* '''Cross-linking-coupled mass spectrometry.'''<ref>{{Cite web|url=http://www.covalx.com/mapping2|title=Epitope Mapping|last=|first=|date=|website=www.covalx.com/epitope2|archive-url=|archive-date=|dead-url=|access-date=2017-02-23}}</ref> Antibody and antigen are bound to a labeled cross-linker, and complex formation is confirmed by high-mass [[Matrix-assisted laser desorption/ionization|MALDI]] detection. The binding location of the antibody to the antigen can then be identified by mass spectrometry (MS). The cross-linked complex is highly stable and can be exposed to various enzymatic and digestion conditions, allowing many different peptide options for detection. MS or [[MS/MS]] techniques are used to detect the amino-acid locations of the labelled cross-linkers and the bound peptides (both [[epitope]] and [[paratope]] are determined in one experiment). The key advantage of this technique is the high sensitivity of MS detection, which means that very little material (hundreds of micrograms or less) is needed.
* '''Cross-linking-coupled mass spectrometry.'''<ref>{{Cite web|url=http://www.covalx.com/mapping2|title=Epitope Mapping|last=|first=|date=|website=www.covalx.com/epitope2|archive-url=|archive-date=|dead-url=|access-date=2017-02-23}}</ref> Antibody and antigen are bound to a labeled cross-linker, and complex formation is confirmed by high-mass [[Matrix-assisted laser desorption/ionization|MALDI]] detection. The binding location of the antibody to the antigen can then be identified by mass spectrometry (MS). The cross-linked complex is highly stable and can be exposed to various enzymatic and digestion conditions, allowing many different peptide options for detection. MS or [[MS/MS]] techniques are used to detect the amino-acid locations of the labelled cross-linkers and the bound peptides (both [[epitope]] and [[paratope]] are determined in one experiment). The key advantage of this technique is the high sensitivity of MS detection, which means that very little material (hundreds of micrograms or less) is needed.

Revision as of 22:39, 3 October 2018

Epitope mapping is the process of experimentally identifying the binding site, or "epitope", of an antibody on its target antigen. Identification and characterization of antibody binding sites aids in the discovery and development of new therapeutics, vaccines, and diagnostics.[1][2] Epitope characterization can also help elucidate the mechanism of binding for an antibody, and experimental epitope mapping data are incorporated into robust algorithms to facilitate in silico prediction of B-cell epitopes based on sequence and/or structural data.[3]

Epitopes are generally divided into two classes: linear and conformational. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are composed of amino acids that are discontinuous in the protein sequence but brought together upon three-dimensional protein folding. Most antigen-antibody interactions rely on binding to conformational epitopes.[4]

Importance for antibody characterization

Epitope mapping for Ebola GP and HER2
Epitope maps obtained by shotgun mutagenesis for antibodies against Ebola virus glycoprotein (GP) and against HER2, an important target in breast cancer. Epitope mapping is important for determining mechanism of action (MOA) and strengthening intellectual property claims.

By providing information on mechanism of action, epitope mapping is a critical component in therapeutic monoclonal antibody (mAb) development. Epitope mapping can reveal how a mAb exerts its functional effects - for instance, by blocking the binding of a ligand or by trapping a protein in a non-functional state.[5] Many therapeutic antibodies target conformational epitopes that are only present when the protein is in its native (properly folded) state, which can make epitope mapping challenging.[4][6] Epitope mapping has been crucial to the development of vaccines against prevalent or deadly viral pathogens, such as chikagunya,[7] dengue,[8] Ebola,[9][10][11] and Zika viruses,[12] by determining the antigenic elements (epitopes) that confer long-lasting immunization effects.[13] 

Complex target antigens, such as membrane proteins (e.g., G protein-coupled receptors [GPCRs]) and multi-subunit proteins (e.g., ion channels) are key targets of drug discovery. Mapping epitopes on these targets can be challenging because of the difficulty in expressing and purifying these complex proteins. Membrane proteins frequently have short antigenic regions (epitopes) that only fold correctly in the context of a lipid bilayer. As a result, mAb epitopes on these membrane proteins are often conformational and, therefore, more difficult to map.[6]

Importance for intellectual property (IP) protection

Epitope mapping has become prevalent in protecting the intellectual property (IP) of therapeutic antibodies. Knowledge of the specific binding sites of antibodies strengthens patents and regulatory submissions by distinguishing between current and prior art (existing antibodies).[14][15] The ability to differentiate accurately between antibodies is particularly important when patenting antibodies against well-validated therapeutic targets that can be drugged by multiple competing antibodies (e.g., PD1 and CD20).[16] In addition to verifying antibody patentability, epitope mapping data have been used to support broad antibody claims submitted to the United States Patent and Trademark Office.[17][18]

Epitope data have been central to several high-profile legal cases involving disputes over the specific protein regions targeted by therapeutic antibodies.[14] In this regard, the Amgen v. Sanofi/Regeneron PCSK9 inhibitor case hinged on the ability to show that both the Amgen and Sanofi/Regeneron therapeutic antibodies bound to overlapping amino acids on the surface of PCSK9.[19]

Methods

There are several methods available for mapping antibody epitopes on target antigens:

  • X-ray co-crystallography. This technique has historically been regarded as the gold-standard approach for epitope mapping because it allows direct visualization of the interaction between the antigen and antibody. However, this approach is technically challenging, time-consuming, and expensive, and it requires large amounts of purified protein. Moreover, not all proteins are amenable to crystallization.[20]
  • Array-based oligo-peptide scanning. Also known as overlapping peptide scan or pepscan analysis, this technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein, and tests for their ability to bind the antibody of interest. This method is fast, relatively inexpensive, and specifically suited to profile epitopes for large numbers of candidate antibodies against a defined target.[13][21] The epitope mapping resolution depends on the number of overlapping peptides that are used. The main disadvantage of this approach is that it cannot generally be used to obtain conformational epitopes, which are the most relevant epitope type for human therapeutic mAbs. However, one study[22] rarely mapped discontinuous epitopes on CD20 using array-based oligo-peptide scanning, by combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (e.g., by using CLIPS scaffolds[23]).
  • Site-directed mutagenesis mapping. The molecular biological technique of site-directed mutagenesis (SDM) can be used to enable epitope mapping. In SDM, systematic mutations of amino acids are introduced into the sequence of the target protein. Binding of an antibody to each mutated protein is tested to identify the amino acids that comprise the epitope. This technique can be used to map both linear and conformational epitopes, but is labor-intensive and time-consuming, typically limiting analysis to a small number of amino-acid residues.[24]
  • High-throughput shotgun mutagenesis mapping.[24][25] The shotgun mutagenesis technique begins with the creation of a mutation library of the entire target antigen, with each clone containing a unique amino acid mutation (typically an alanine-to-serine substitution). Hundreds of plasmid clones from the library are individually arrayed in 384-well micro plates, expressed in mammalian cells, and tested for antibody binding. Amino acids of the target required for antibody binding are identified by a loss of immunoreactivity. These residues are mapped onto structures of the target protein to visualize the epitope. Benefits of high-throughput mutagenesis mapping include: 1) the ability to identify both linear and conformational epitopes, 2) in a fraction of the time required by other methods, 3) the presentation of properly folded and post-translationally modified proteins, and 4) the ability to identify key amino acids that drive the energetic interactions (energetic "hot spots" of the epitope)[26][27]. High-throughput shotgun mutagenesis mapping is a well-validated and widely used approach that has been used to obtain single-amino-acid-resolution epitopes for over 1,000 mAbs,[28] revealing mechanistic information for protein-drug interactions,[29][30] the functional regions of complex membrane proteins, including GPCRs,[31][32] target binding information for small molecules,[33][34] and epitope information for IP protection purposes.[17][18]
  • Hydrogen–deuterium exchange. This method, which is growing in popularity, gives information about the solvent accessibility of various parts of the antigen and antibody, demonstrating reduced solvent accessibility in regions of protein-protein interactions.[35]
  • Cross-linking-coupled mass spectrometry.[36] Antibody and antigen are bound to a labeled cross-linker, and complex formation is confirmed by high-mass MALDI detection. The binding location of the antibody to the antigen can then be identified by mass spectrometry (MS). The cross-linked complex is highly stable and can be exposed to various enzymatic and digestion conditions, allowing many different peptide options for detection. MS or MS/MS techniques are used to detect the amino-acid locations of the labelled cross-linkers and the bound peptides (both epitope and paratope are determined in one experiment). The key advantage of this technique is the high sensitivity of MS detection, which means that very little material (hundreds of micrograms or less) is needed.

Other methods, such as phage display and limited proteolysis, provide high-throughput monitoring of antibody binding but lack reliability, especially for conformational epitopes.[4]

See also

References

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External links